This project is focused on understanding clathrin-independent forms of endocytosis. Endocytosis that occurs without clathrin coats occurs in all cells but is poorly understood. We are interested in studying the cargo proteins that enter cells by this mechanism, their intracellular itinerary once they have been internalized and whether they contain amino acid sequences that allow for specialized sorting within cells. We have been identifying new cargo proteins and found that a subset of these proteins take alternative traffic routes once they have entered cells. The major histocompatibility complex Class I protein (MHCI), is a prototypical clathrin-indepenent cargo protein and after internalization it reaches endosomes that contain cargo proteins such as the transferrin receptor that enter via clathrin-depenent endocytosis. From there, MHCI travels either to late endosomes and lysosomes where it is degraded or on to recycling tubules that return MHCI back to the cell surface. CD44, CD98, and CD147, however, show an altered itinerary in many cells where they traffic directly into the recycling tubules and avoid trafficking to late endosomal compartments. Consistent with this altered itinerary, CD44, CD98 and CD147 are long-lived proteins and are not degraded like MHCI, which is routed to late endosomes. With a goal to understanding how PM proteins entering cells by clathrin-independent endocytosis are targeted to degradation in lysosomes, we examined the effect of expression of MARCH family of E-3 ligases. We found that expression of MARCH8 dramatically altered the trafficking of CD44 and CD98, causing these proteins to enter cells and be trafficked to late endosomes for degradation. CD98 was directly ubiquitinylated by MARCH8 and the resultant trafficking to lysosomes resulted in a substantial downregulation of surface CD98. Intriguingly although both CD44 and CD98 were affected by expression of MARCH8, CD98, and not CD44, was downregulated by a closely related MARCH, MARCH1. This suggests that the MARCHE3 ligases exhibit distinct substrate specificities, that we are currently trying to dissect.